D27 Antibody

What is the D27 antibody and how was it initially developed?

D27 is a neutralizing antibody (nAb) initially developed through computational antibody design using the Rosetta software suite. In its original form, D27 exhibited relatively low binding affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 Wuhan-Hu-1 strain . The antibody was computationally designed as part of efforts to develop broadly neutralizing antibodies against coronaviruses, with the initial design serving as a foundation for further optimizations . The crystal structure of D27-Fab bound to SARS-CoV-2 RBD was determined at 2.2 Å resolution (PDB ID: 7VYR), which validated that the computational design was largely correct .

How did D27 evolve into more potent derivatives?

The evolution from D27 to more potent derivatives followed a stepwise optimization process:

• Starting point: D27 antibody with low binding affinity to the wild-type SARS-CoV-2 RBD

• First optimization: D27LE was developed by extending the complementary determining regions 3 (CDR3) at the tips by 1 or 2 residues and additionally randomizing the flanking residues

• Final optimization: D27LEY was created through computational sequence optimization of four residues near the Y501 position of the SARS-CoV-2 RBD

This systematic approach demonstrates how structure-based computational design can be used to progressively improve antibody affinity and breadth of neutralization .

What is the significance of the N501Y mutation in relation to D27LEY?

The N501Y mutation is present in more than 80% of SARS-CoV-2 variants, including Alpha and Omicron. This mutation is considered adaptive, enabling tighter interaction with the human ACE2 receptor . D27LEY was intentionally optimized as an "N501Y-centric antibody" to target this mutation specifically, which explains its extremely high binding affinity (picomolar range) for variants containing the N501Y mutation .

Despite being optimized for Y501, D27LEY remarkably maintains tight binding (KD < 1 nM) to RBDs with different amino acids at position 501, demonstrating its versatility across variants . The crystal structure reveals that the Y501-containing loop adopts a ribbon-like topology and serves as a small but major epitope in which Y501 is part of extensive intermolecular interactions .

How does the binding affinity of D27LEY compare across different SARS-CoV-2 variants?

D27LEY exhibits varying binding affinities across different SARS-CoV-2 variants, as measured by biolayer interferometry (BLI). The following table summarizes these differences:

The binding affinity pattern indicates that while D27LEY was optimized for the N501Y mutation, it maintains remarkable cross-reactivity across variants with different mutations .

What is the structural basis for D27LEY's broad cross-reactivity?

The crystal structure of D27LEY-Fab in complex with the RBD of the Alpha variant provides critical insights into its broad cross-reactivity. Key structural features include:

• The epitope recognized by D27LEY is highly conserved across sarbecoviruses, with N501 being the only variable residue among its epitope residues in SARS-CoV-2 variants

• D27LEY targets an antigenic site on the RBD that partly overlaps with site IIa, which is evolutionarily conserved across sarbecoviruses

• Unlike antibodies that bind to the receptor binding motif (RBM), which contains highly variable regions, D27LEY's binding surface is focused on a more conserved region of the RBD

• The Y501-containing loop adopts a ribbon-like topology that serves as a small but major epitope with extensive intermolecular interactions

• D27LEY's binding surface is closer to the RBM than other broadly neutralizing antibodies, spanning a wider patch on the RBD

This strategic targeting of conserved epitopes explains why D27LEY maintains effectiveness despite mutations at other sites in the RBD .

How does neutralization potency correlate with binding affinity for D27LEY?

In vitro neutralization studies reveal a strong correlation between binding affinity and neutralization potency for D27LEY across different SARS-CoV-2 variants:

*GMT: Geometric Mean Titer from VSV pseudotyped neutralization assay

These data demonstrate that neutralization potency generally correlates with binding affinity, with some variations likely due to differences in infection efficiency between variants . Despite the Delta variant having stronger binding affinity than wild-type, its neutralization constant is slightly higher, which may reflect Delta's enhanced infectivity .

What experimental techniques are optimal for measuring D27LEY binding to coronavirus RBDs?

Based on the research with D27LEY, several methodological approaches have proven effective:

• Biolayer Interferometry (BLI): The Octet R8 system (Sartorius) was used effectively to measure binding kinetics. For optimal results:

  • Load biotinylated SARS-CoV-2 variant RBDs at 5 nM onto streptavidin biosensor tips

  • For His-tagged RBDs (e.g., SARS-CoV-1), use Ni-NTA biosensor tips

  • Immerse in Kinetics Buffer for 120 seconds

  • Test antibodies at five different concentrations with association (240-360s) and dissociation (720s) steps

  • Analyze binding kinetics with Octet BLI Analysis software

• Neutralization Assays:

  • Vesicular stomatitis virus (VSV) pseudotyped neutralization assay for initial screening

  • Focus reduction neutralization test (FRNT) in Vero E6 cells for more precise neutralization constants

• Structural Analysis:

  • X-ray crystallography to determine complex structures (as done for D27-Fab–WT RBD and D27LEY-Fab–Alpha RBD)

  • Resolution of 2.2 Å is sufficient to observe key interactions

These methodologies provide complementary data on binding affinity, neutralization potency, and structural determinants of cross-reactivity .

How can computational approaches be used to further optimize D27LEY or design similar broadly neutralizing antibodies?

The successful development of D27LEY provides a blueprint for computational antibody design:

• Initial Design Phase:

  • Use Rosetta software suite for antibody scaffold design

  • Focus on targeting conserved epitopes rather than highly variable regions

  • Validate initial designs through structural studies

• Optimization Strategies:

  • CDR extension and randomization: Extend CDR3 loops by 1-2 residues and randomize flanking residues

  • Epitope-focused optimization: Identify key residues on the target (e.g., N501Y) and optimize neighboring antibody residues

  • "Hot spot" targeting: Create extended CDR loops that can establish strong interactions with conserved regions

• Validation Methods:

  • Iterative binding assays to confirm improvements in affinity

  • Structural studies to validate design principles

  • Cross-reactivity testing against multiple variants and related viruses

• Future Directions:

  • Use D27LEY's binding mode as a template for designing pan-sarbecovirus vaccines

  • Focus on the conserved epitope identified through D27LEY's structural studies

  • Implement similar computational approaches for other viral targets

This methodology demonstrates how computational design can address viral escape through rational targeting of conserved epitopes .

What challenges might researchers encounter when studying D27LEY's effectiveness against emerging variants?

Researchers studying D27LEY against emerging variants should anticipate several challenges:

• Epitope Evolution:

  • While D27LEY targets a conserved region, continued viral evolution could eventually lead to mutations in this epitope

  • Regular structural monitoring of new variants is necessary to assess potential impacts on binding

• Methodological Considerations:

  • For variants with significantly altered RBD structures, standard BLI protocols may need adjustment

  • Pseudovirus neutralization assays may not fully recapitulate the behavior of authentic virus

  • Live virus neutralization requires enhanced biosafety measures

• Expression Systems:

  • Different expression systems for recombinant RBDs may introduce post-translational modifications that affect binding

  • Standardization across studies is important for comparative analyses

• In Vivo Translation:

  • In vitro binding and neutralization may not perfectly predict in vivo efficacy

  • Animal models should be selected carefully to represent human ACE2 interactions

Researchers should implement regular monitoring of emerging variants and be prepared to adapt methodological approaches accordingly .